System 1: Widefield microscope for single and dual wavelength fluorescence measurements in live cells.
Zeiss Axiovert 10 epifluorescence microscope (inverted) with 40x long-working distance and 63x oil immersion objectives. Interfaced with Atto Biosciences intensified CCD camera (8 bit, 512 x 480 pixels), camera controller, filter changer, high speed shutter and RatioVision software. Temperature-controlled superfusion chamber (accepts 18 or 22 mm round glass coverslips) and associated pumps, etc. Stand alone analysis software (PC only).
This system is designed for [ion]i measurements in live cells using single excitation (e.g. fluo-3 for [Ca2+]i) or dual excitation (e.g. fura-2 for [Ca2+]i, BCECF for pHi, SBFI for [Na+]i, PBFI for [K+]i) probes. Although very sensitive, the camera on this system has limited spatial resolution (reliable recordings can be obtained only from structures and/or cells >5 µm diam.).
System 2: Widefield microscope for single wavelength, dual wavelength and dual emission fluorescence measurements in live cells.
Zeiss Axiovert 135 epifluorescence microscope (inverted) with 40x long-working distance and 63x oil immersion objectives. Interfaced with two Atto Biosciences intensified CCD cameras (each 8 bit, 512 x 480 pixels), camera controller, filter changer, high speed shutter and RatioVision software. Temperature-controlled superfusion chamber (accepts 18 or 22 mm round glass coverslips) and associated pumps, etc. Stand alone analysis software (PC only).
This system can perform the same [ion]i measurements (and has the same limited spatial resolution) as System #1. The addition of a second camera and filters allows the use of dual emission (e.g. indo for [Ca2+], SNARF for pH) as well as dual excitation probes. A particular advantage of this system is the ability to measure the intracellular concentrations of more than one ionic species simultaneously (e.g. Sheldon et al., J. Neurosci. 24, 11057-11069, 2004). Automated routines are available for the simultaneous measurement of [Na+]i or [K+]i + pHi; [Ca2+]i + pHi; [Cl-]i + pHi; [Na+]i or [K+]i + [Ca2+]i; [Ca2+]i + [cAMP]i; and [Ca2+]i + mitochondrial membrane potential (e.g. Fernandes et al., J. Neurosci. 27, 13614-13623, 2007). Other routines are easily developed and electrophysiological recordings can be performed simultaneously with dual [ion]i measurements (e.g. Kelly & Church, J. Neurophysiol. 96, 2342-2353, 2006).
System 3: Widefield microscope for single and dual wavelength fluorescence measurements in live cells.
Zeiss Axioskop 2 FSPlus microscope (upright) with 20x, 40x, 63x and 100x water-dipping objectives. Interfaced with a QImaging Retiga EXi cooled CCD camera (12 bit, 1360 x 1036 pixels), Sutter DG5 high speed filter changer and shutter, and Intelligent Imaging Innovations software (Slidebook). Temperature-controlled superfusion chamber (accepts 15 mm round glass coverslips) and associated pumps, etc. Stand alone analysis software (PC only).
This system can perform the same [ion]i measurements as System #1, with the added advantage of high spatial resolution. DIC images can be captured during the course of ratiometric [ion]i measurements, making this system particularly useful for examining changes in [ion]i at the leading edge of migrating cells or neuronal growth cones.
